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Therapy-induced tumour secretomes promote resistance and tumour progression.

Obenauf AC, Zou Y, Ji AL, Vanharanta S, Shu W, Shi H, Kong X, Bosenberg MC, Wiesner T, Rosen N, Lo RS, Massagué J - Nature (2015)

Bottom Line: Drug resistance invariably limits the clinical efficacy of targeted therapy with kinase inhibitors against cancer.Here we show that targeted therapy with BRAF, ALK or EGFR kinase inhibitors induces a complex network of secreted signals in drug-stressed human and mouse melanoma and human lung adenocarcinoma cells.The tumour-promoting secretome of melanoma cells treated with the kinase inhibitor vemurafenib is driven by downregulation of the transcription factor FRA1.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.

ABSTRACT
Drug resistance invariably limits the clinical efficacy of targeted therapy with kinase inhibitors against cancer. Here we show that targeted therapy with BRAF, ALK or EGFR kinase inhibitors induces a complex network of secreted signals in drug-stressed human and mouse melanoma and human lung adenocarcinoma cells. This therapy-induced secretome stimulates the outgrowth, dissemination and metastasis of drug-resistant cancer cell clones and supports the survival of drug-sensitive cancer cells, contributing to incomplete tumour regression. The tumour-promoting secretome of melanoma cells treated with the kinase inhibitor vemurafenib is driven by downregulation of the transcription factor FRA1. In situ transcriptome analysis of drug-resistant melanoma cells responding to the regressing tumour microenvironment revealed hyperactivation of several signalling pathways, most prominently the AKT pathway. Dual inhibition of RAF and the PI(3)K/AKT/mTOR intracellular signalling pathways blunted the outgrowth of the drug-resistant cell population in BRAF mutant human melanoma, suggesting this combination therapy as a strategy against tumour relapse. Thus, therapeutic inhibition of oncogenic drivers induces vast secretome changes in drug-sensitive cancer cells, paradoxically establishing a tumour microenvironment that supports the expansion of drug-resistant clones, but is susceptible to combination therapy.

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The regressing tumour microenvironment stimulates the outgrowth, infiltration and metastasis of drug-resistant clonesa, Schematic of the experimental setup. b, Bioluminescent signal of drug-resistant A375R-TGL cells in vemurafenib-sensitive, A375 tumours, treated with vehicle or vemurafenib for 5 days (vehicle, n = 36; vemurafenib, n = 15 tumours). c, EdU incorporation in A375R-TGL cells in A375/A375R-TGL tumours treated with vehicle or vemurafenib for 4 days, as determined by FACS (vehicle, n = 8; vemurafenib, n = 6 tumours). d, Bioluminescent signal of A375R-TGL tumours alone, treated with vehicle or vemurafenib for 5 days (vehicle, n = 38; vemurafenib, n = 15 tumours). e, Bioluminescent signal of TGL-expressing drug-resistant cancer cells (A375R, M249R4, PC9, H2030) in drug-sensitive tumours (Colo800, LOX, UACC62, M249, H3122, HCC827) treated with vehicle or drugs (vemurafenib, crizotinib, erlotinib) for 5 days (n (from left to right on the graph, in this order) = 6, 7, 12, 12, 9, 9, 25, 26, 9, 12, 12, 12, 16, 11 tumours). f, Spontaneous lung metastasis by A375R cells in mice bearing A375/A375R-TGL tumours treated with vehicle or vemurafenib (10 days), visualized by BLI (n = 4). g, Seeding of A375R-TGL cells from the circulation to unlabelled, subcutaneous A375 tumours of mice treated with vehicle or vemurafenib. Signal in the tumour was quantified by BLI (vehicle, n = 30; vemurafenib, n = 34 tumours; three independent experiments combined). h, Treatment response, determined by tumour size, of subcutaneous A375 tumours allowed to be seeded by A375R−TGL cells from the circulation or mock injected (vehicle, n = 16; vemurafenib, n = 8 tumours). Data in b–e,g,h, are average; error bars represent s.e.m; data in f, center line is median, whiskers are min. to max. P values shown were calculated using a two-tailed Mann-Whitney test (* p<0.05, ** p<0.01, *** p<0.001, n.s.= not significant).
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Figure 1: The regressing tumour microenvironment stimulates the outgrowth, infiltration and metastasis of drug-resistant clonesa, Schematic of the experimental setup. b, Bioluminescent signal of drug-resistant A375R-TGL cells in vemurafenib-sensitive, A375 tumours, treated with vehicle or vemurafenib for 5 days (vehicle, n = 36; vemurafenib, n = 15 tumours). c, EdU incorporation in A375R-TGL cells in A375/A375R-TGL tumours treated with vehicle or vemurafenib for 4 days, as determined by FACS (vehicle, n = 8; vemurafenib, n = 6 tumours). d, Bioluminescent signal of A375R-TGL tumours alone, treated with vehicle or vemurafenib for 5 days (vehicle, n = 38; vemurafenib, n = 15 tumours). e, Bioluminescent signal of TGL-expressing drug-resistant cancer cells (A375R, M249R4, PC9, H2030) in drug-sensitive tumours (Colo800, LOX, UACC62, M249, H3122, HCC827) treated with vehicle or drugs (vemurafenib, crizotinib, erlotinib) for 5 days (n (from left to right on the graph, in this order) = 6, 7, 12, 12, 9, 9, 25, 26, 9, 12, 12, 12, 16, 11 tumours). f, Spontaneous lung metastasis by A375R cells in mice bearing A375/A375R-TGL tumours treated with vehicle or vemurafenib (10 days), visualized by BLI (n = 4). g, Seeding of A375R-TGL cells from the circulation to unlabelled, subcutaneous A375 tumours of mice treated with vehicle or vemurafenib. Signal in the tumour was quantified by BLI (vehicle, n = 30; vemurafenib, n = 34 tumours; three independent experiments combined). h, Treatment response, determined by tumour size, of subcutaneous A375 tumours allowed to be seeded by A375R−TGL cells from the circulation or mock injected (vehicle, n = 16; vemurafenib, n = 8 tumours). Data in b–e,g,h, are average; error bars represent s.e.m; data in f, center line is median, whiskers are min. to max. P values shown were calculated using a two-tailed Mann-Whitney test (* p<0.05, ** p<0.01, *** p<0.001, n.s.= not significant).

Mentions: In order to model therapeutic targeting of heterogeneous tumour cell populations in vivo, we mixed a small percentage of vemurafenib-resistant A375 human melanoma cells (A375R), labelled with a TK-GFP-Luciferase vector (TGL), together with a majority of non-labelled, vemurafenib-sensitive A375 cells, and injected the admixture (A375/A375R, 99.95/0.05%) subcutaneously in mice (Extended Data Fig. 1a). After the tumours were established, we treated the mice with vemurafenib or vehicle, and monitored the growth of resistant cells by bioluminescent imaging (BLI) in vivo (Fig. 1a). While vemurafenib treatment decreased the volume of sensitive tumours (A375 alone) (Extended Data Fig. 1b), the number of admixed resistant cells in regressing tumours (A375/A375R) significantly increased compared to vehicle-treated controls (Fig. 1b). GFP staining confirmed increased numbers of resistant cells in regressing tumours, and EdU or BrdU staining confirmed their increased proliferation rate compared to the vehicle treated controls (Fig. 1c, Extended Data Fig. 1c, d). Tumours comprised of only resistant cells showed no growth difference when treated with vehicle or vemurafenib (Fig. 1d), indicating that the growth advantage of resistant cells in regressing tumours was not caused by direct effects of vemurafenib on cancer or stromal cells.


Therapy-induced tumour secretomes promote resistance and tumour progression.

Obenauf AC, Zou Y, Ji AL, Vanharanta S, Shu W, Shi H, Kong X, Bosenberg MC, Wiesner T, Rosen N, Lo RS, Massagué J - Nature (2015)

The regressing tumour microenvironment stimulates the outgrowth, infiltration and metastasis of drug-resistant clonesa, Schematic of the experimental setup. b, Bioluminescent signal of drug-resistant A375R-TGL cells in vemurafenib-sensitive, A375 tumours, treated with vehicle or vemurafenib for 5 days (vehicle, n = 36; vemurafenib, n = 15 tumours). c, EdU incorporation in A375R-TGL cells in A375/A375R-TGL tumours treated with vehicle or vemurafenib for 4 days, as determined by FACS (vehicle, n = 8; vemurafenib, n = 6 tumours). d, Bioluminescent signal of A375R-TGL tumours alone, treated with vehicle or vemurafenib for 5 days (vehicle, n = 38; vemurafenib, n = 15 tumours). e, Bioluminescent signal of TGL-expressing drug-resistant cancer cells (A375R, M249R4, PC9, H2030) in drug-sensitive tumours (Colo800, LOX, UACC62, M249, H3122, HCC827) treated with vehicle or drugs (vemurafenib, crizotinib, erlotinib) for 5 days (n (from left to right on the graph, in this order) = 6, 7, 12, 12, 9, 9, 25, 26, 9, 12, 12, 12, 16, 11 tumours). f, Spontaneous lung metastasis by A375R cells in mice bearing A375/A375R-TGL tumours treated with vehicle or vemurafenib (10 days), visualized by BLI (n = 4). g, Seeding of A375R-TGL cells from the circulation to unlabelled, subcutaneous A375 tumours of mice treated with vehicle or vemurafenib. Signal in the tumour was quantified by BLI (vehicle, n = 30; vemurafenib, n = 34 tumours; three independent experiments combined). h, Treatment response, determined by tumour size, of subcutaneous A375 tumours allowed to be seeded by A375R−TGL cells from the circulation or mock injected (vehicle, n = 16; vemurafenib, n = 8 tumours). Data in b–e,g,h, are average; error bars represent s.e.m; data in f, center line is median, whiskers are min. to max. P values shown were calculated using a two-tailed Mann-Whitney test (* p<0.05, ** p<0.01, *** p<0.001, n.s.= not significant).
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Related In: Results  -  Collection

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Figure 1: The regressing tumour microenvironment stimulates the outgrowth, infiltration and metastasis of drug-resistant clonesa, Schematic of the experimental setup. b, Bioluminescent signal of drug-resistant A375R-TGL cells in vemurafenib-sensitive, A375 tumours, treated with vehicle or vemurafenib for 5 days (vehicle, n = 36; vemurafenib, n = 15 tumours). c, EdU incorporation in A375R-TGL cells in A375/A375R-TGL tumours treated with vehicle or vemurafenib for 4 days, as determined by FACS (vehicle, n = 8; vemurafenib, n = 6 tumours). d, Bioluminescent signal of A375R-TGL tumours alone, treated with vehicle or vemurafenib for 5 days (vehicle, n = 38; vemurafenib, n = 15 tumours). e, Bioluminescent signal of TGL-expressing drug-resistant cancer cells (A375R, M249R4, PC9, H2030) in drug-sensitive tumours (Colo800, LOX, UACC62, M249, H3122, HCC827) treated with vehicle or drugs (vemurafenib, crizotinib, erlotinib) for 5 days (n (from left to right on the graph, in this order) = 6, 7, 12, 12, 9, 9, 25, 26, 9, 12, 12, 12, 16, 11 tumours). f, Spontaneous lung metastasis by A375R cells in mice bearing A375/A375R-TGL tumours treated with vehicle or vemurafenib (10 days), visualized by BLI (n = 4). g, Seeding of A375R-TGL cells from the circulation to unlabelled, subcutaneous A375 tumours of mice treated with vehicle or vemurafenib. Signal in the tumour was quantified by BLI (vehicle, n = 30; vemurafenib, n = 34 tumours; three independent experiments combined). h, Treatment response, determined by tumour size, of subcutaneous A375 tumours allowed to be seeded by A375R−TGL cells from the circulation or mock injected (vehicle, n = 16; vemurafenib, n = 8 tumours). Data in b–e,g,h, are average; error bars represent s.e.m; data in f, center line is median, whiskers are min. to max. P values shown were calculated using a two-tailed Mann-Whitney test (* p<0.05, ** p<0.01, *** p<0.001, n.s.= not significant).
Mentions: In order to model therapeutic targeting of heterogeneous tumour cell populations in vivo, we mixed a small percentage of vemurafenib-resistant A375 human melanoma cells (A375R), labelled with a TK-GFP-Luciferase vector (TGL), together with a majority of non-labelled, vemurafenib-sensitive A375 cells, and injected the admixture (A375/A375R, 99.95/0.05%) subcutaneously in mice (Extended Data Fig. 1a). After the tumours were established, we treated the mice with vemurafenib or vehicle, and monitored the growth of resistant cells by bioluminescent imaging (BLI) in vivo (Fig. 1a). While vemurafenib treatment decreased the volume of sensitive tumours (A375 alone) (Extended Data Fig. 1b), the number of admixed resistant cells in regressing tumours (A375/A375R) significantly increased compared to vehicle-treated controls (Fig. 1b). GFP staining confirmed increased numbers of resistant cells in regressing tumours, and EdU or BrdU staining confirmed their increased proliferation rate compared to the vehicle treated controls (Fig. 1c, Extended Data Fig. 1c, d). Tumours comprised of only resistant cells showed no growth difference when treated with vehicle or vemurafenib (Fig. 1d), indicating that the growth advantage of resistant cells in regressing tumours was not caused by direct effects of vemurafenib on cancer or stromal cells.

Bottom Line: Drug resistance invariably limits the clinical efficacy of targeted therapy with kinase inhibitors against cancer.Here we show that targeted therapy with BRAF, ALK or EGFR kinase inhibitors induces a complex network of secreted signals in drug-stressed human and mouse melanoma and human lung adenocarcinoma cells.The tumour-promoting secretome of melanoma cells treated with the kinase inhibitor vemurafenib is driven by downregulation of the transcription factor FRA1.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.

ABSTRACT
Drug resistance invariably limits the clinical efficacy of targeted therapy with kinase inhibitors against cancer. Here we show that targeted therapy with BRAF, ALK or EGFR kinase inhibitors induces a complex network of secreted signals in drug-stressed human and mouse melanoma and human lung adenocarcinoma cells. This therapy-induced secretome stimulates the outgrowth, dissemination and metastasis of drug-resistant cancer cell clones and supports the survival of drug-sensitive cancer cells, contributing to incomplete tumour regression. The tumour-promoting secretome of melanoma cells treated with the kinase inhibitor vemurafenib is driven by downregulation of the transcription factor FRA1. In situ transcriptome analysis of drug-resistant melanoma cells responding to the regressing tumour microenvironment revealed hyperactivation of several signalling pathways, most prominently the AKT pathway. Dual inhibition of RAF and the PI(3)K/AKT/mTOR intracellular signalling pathways blunted the outgrowth of the drug-resistant cell population in BRAF mutant human melanoma, suggesting this combination therapy as a strategy against tumour relapse. Thus, therapeutic inhibition of oncogenic drivers induces vast secretome changes in drug-sensitive cancer cells, paradoxically establishing a tumour microenvironment that supports the expansion of drug-resistant clones, but is susceptible to combination therapy.

Show MeSH
Related in: MedlinePlus